Abstract
We have inactivated the genes encoding components of MntABC, an ABC (ATP binding cassette) transporter system for manganese in the cyanobacterium Synechocystis sp. PCC 6803. The growth rates of these mutant strains were significantly lower in a manganese-deficient medium and were restored to near normal levels upon addition of micromolar concentrations of Mn2+, indicating the presence of a second transport system for manganese in this organism. 54Mn2+ uptake experiments indicated that the MntABC transporter was induced under manganese starvation conditions, whereas the second transporter system was induced in the presence of micromolar levels of manganese. Both of these systems were nonfunctional at low temperatures and could transport trace levels of 54Mn2+, reflecting high affinity active transport. The initial rates of Mn2+ uptake for cells grown with or without manganese exhibited biphasic saturation kinetics, suggesting that Mn2+ can also be accumulated by a low affinity system in these bacteria. The kinetic parameters for the MntABC transporter system are Km = 1-3 microM and Vmax = 3-8 pmol/min.10(8) cells. Accumulation of manganese by this system was competitively inhibited by Cd2+ (Ki = 4-8 microM), Co2+ and Zn2+ (Ki = 8-15 microM). In contrast, the second high affinity system was highly specific for manganese and was not inhibited by any tested metal ion. We have also demonstrated that in this organism, photosynthetic electron transport is necessary for optimal rates of manganese uptake.
Highlights
CAB encodes the components of an ABC (ATP binding cassette)-type transporter system, the first such protein complex for manganese identified in any organism
These results indicated that either the mutation in the mntA gene in the BP13 strain does not completely inhibit the function of the MntABC transporter system, or manganese uptake can be mediated by additional transporters that function under higher levels of manganese
One band of expected size was observed in each mutant strain, indicating that the kanamycin resistance (Kmr)-gene cartridge was inserted in the genome of each mutant strain, and each of the mutations was completely segregated
Summary
The Synechocystis 6803 strains were grown at 30 °C under 60 microeinsteins1⁄7mϪ2 sϪ1 of white light in the BG11 medium [4], for which MnCl2 was sterilized separately from other components and added to a final concentration of 5 M. Mn2ϩ Uptake Assay—For Mn2ϩ uptake experiments, Synechocystis 6803 cells, usually maintained on solid BG11 medium, were inoculated into liquid BG11-Mn medium and grown for 30 –36 h. These cells were harvested by centrifugation, washed, and resuspended in fresh BG11-Mn with the addition of different concentrations of MnCl2 where indicated. The filters were suspended in scintillation mixture and counted on an LS 5000 TD
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